EDUCATIONAL COMMENTARY DIFFERENTIATING IMMATURE PERIPHERAL BLOOD CELLS

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1 Educational commentary is provided through our affiliation with the American Society for Clinical Pathology (ASCP). To obtain FREE CME/CMLE credits click on Continuing Education on the left side of the screen. Learning Outcomes Upon completion of this exercise, participants will be able to: describe the characteristic morphology of normal peripheral blood leukocytes. identify the morphologic features of immature neutrophils. differentiate blood cells with similar morphologic characteristics. Case Example A 42-year-old woman went to her doctor and reported excessive bruising. Laboratory findings included a white blood cell count of /µl ( /L), a red blood cell (RBC) count of /µl ( /L), a hemoglobin level of 8.5 g/dl (85 g/l), a hematocrit of 25%, a platelet count of /µl ( /L), a mean corpuscular volume of 87.6 fl, a mean corpuscular hemoglobin of 31.8 pg, a mean corpuscular hemoglobin concentration of 34.0 g/dl (340 g/l), and a red blood cell distribution width of 13.5%. She was diagnosed with acute myelocytic (myeloblastic) leukemia. The images provided are both normal and abnormal cells that may be seen in the peripheral blood in this condition. Commentary Image BCI-08 illustrates a nucleated RBC. A nucleated RBC is a young erythrocyte that has not yet matured and still retains its nucleus. Nucleated RBCs are normally seen in bone marrow samples but not in the peripheral blood; sometimes they may be present in the peripheral blood of newborns and infants as a normal finding. The presence of nucleated RBCs in the peripheral blood of an adult suggests abnormally accelerated erythropoiesis. Nucleated RBCs represent specific stages in erythrocyte maturation but do not need to be classified when seen on a peripheral blood smear. However, they should be counted and reported. The one in this image is typical of those seen when nucleated erythrocytes are present. Note the dense and clumped nucleus with a blue-gray cytoplasm. Editor s note: Some participants reported a plasma cell for Sample BCI-08. When evaluating cells, it is important to take into consideration cytoplasmic features and nuclear characteristics. Plasma cells

2 typically have a deep/dark blue cytoplasm (the cytoplasm in this NRBC is gray-blue and not the nice skyblue of plasma cells). Any NRBC with a deep blue cytoplasm would also be larger and have nuclear chromatin that is more loose and open (a definitely immature cell). Plasma cells also have a more prominent Golgi area adjacent to the nucleus. While the NRBC in this image appears to have a Golgi, it extends to the cytoplasmic margin and is not clearly defined (in contrast to the Golgi in a plasma cell that will be localized adjacent to the nucleus and is clearly visible). Finally, plasma cells are mature cells, so the nuclear chromatin is dense and clumped, sometimes even appearing like a spoke-wheel ; no nucleoli are evident. Image BCI-09 is an example of a small, normal lymphocyte. Lymphocytes vary in size. In a small lymphocyte, the nucleus occupies almost the entire cell; just a scanty rim of blue cytoplasm can be seen. The nuclei are usually oval, round, or slightly indented. The nuclear chromatin is condensed, clumped, and dark purple in color. Sometimes nucleoli may be present, as in this cell. However, the dense chromatin indicates that this is a mature cell. The nucleoli-like areas are not prominent. Small lymphocytes may be confused with nucleated RBCs, as seen in Image BCI-08. Note that the appearance of the chromatin in the nucleated RBC is dense and almost featureless. Though the nucleus in the lymphocyte is clumped, light and dark staining areas are still visible. Nucleated erythrocytes may also have more cytoplasm than a small lymphocyte, but the cytoplasm may be more blue-gray or a faded blue color. In contrast, the cytoplasm in a small lymphocyte is a more typical blue. Image BCI-10 shows a monocyte. Monocytes are the largest cells that are normally seen in the peripheral blood. The cytoplasm of a monocyte is usually abundant, vacuolated, and blue-gray in color. It may appear uneven or rough. Cytoplasmic projections are sometimes visible, although they are not present in this example. This cell has typical cytoplasmic vacuoles. Monocytic nuclei are variable in shape and may be round, oval, kidney-shaped, or lobulated. The nuclear chromatin is generally fine,

3 has minimal clumping, and stains a lighter purple or pink. Image BCI-11 is a segmented neutrophil. Neutrophils typically have 2 to 5 nuclear lobes connected by thin strands of chromatin. The chromatin is condensed and clumped. The cytoplasm of neutrophils has numerous small pink, violet-pink, or tan granules. The individual cytoplasmic granules in segmented neutrophils may be hard to differentiate, but their presence contributes to an overall pink color and grainy appearance. The arrow in Image BCI-12 points to a blast cell. Blast cells should not be present in the peripheral blood. They are typically large cells and have a high nuclear to cytoplasmic ratio. The nucleus is round or oval with loose, open chromatin. Multiple prominent nucleoli may be visible, as in this example. The cytoplasm in blasts is usually very blue and contains no granules. Because blasts of different cell lines are similar morphologically, it is not always possible to classify the cells according to lineage. Blasts should be identified and reported, and additional techniques are used to determine the lineage of the cells. Note that blasts can be identified in other images, including Images BCI-09, BCI-10, BCI-11, and BCI-12. The differential cell count for this patient reported 69% blasts. Blasts should not be confused with atypical (reactive) lymphocytes. Atypical lymphocytes are most often associated with viral illnesses such as infectious mononucleosis. While atypical lymphocytes and blasts share similar morphologic features, including large size, important differences exist. Atypical lymphocytes are reacting or responding to an infection; therefore, a variety of morphologic appearances can be seen, reflecting the heterogeneity of the immune response. Blast cells originate from a malignant, monoclonal proliferation, and, therefore, the cells appear more homogeneous. While atypical lymphocytes may have dark blue cytoplasm, some cells will have cytoplasm that is gray or pale blue.

4 Likewise, the nuclei in reactive lymphocytes may be round, but they may also be oval, folded, or indented. The nuclear chromatin may be fine or moderately to coarsely clumped. Nucleoli are not usually visible. Note that all the blasts seen in this patient are similar in morphology. The nuclei are generally oval, the chromatin is loose and open, prominent nucleoli are present, and there is a moderate amount of deep blue cytoplasm. Image BCI-13 features a myelocyte. As with nucleated RBCs and blasts, myelocytes are not normally seen in the peripheral blood. Classic myelocytes have a nucleus that is eccentrically located and is usually oval or round; nucleoli are usually not visible, though one may be present in this image. The chromatin is beginning to clump, but lighter areas of parachromatin may still be seen. Many times a clear area, or hof, is visible adjacent to the nucleus. The myelocyte is the last stage of the neutrophil maturation sequence capable of mitosis, but the first in which the violet, tan, or pink specific granules are seen. The darker purple nonspecific or primary granules frequently are still present, as in this cell. The arrow in Image BCI-14 points to a metamyelocyte. Metamyelocytes are also not normally seen in the peripheral blood. They feature a nucleus that is slightly indented or shaped like a kidney bean. The nuclear chromatin is clumped and dense. However, some loose chromatin reflects the immaturity of this cell. Nucleoli are not present. Note the lighter staining areas that are too undefined to represent nucleoli; they may be staining artifact. Generally the cytoplasm in metamyelocytes has violet, tan, or pink specific granules, although this cell has retained some blue cytoplasm. Summary Images BCI-09 through BCI-14 show peripheral blood cells from a patient with acute myelocytic (myeloblastic) leukemia. Although some normal cells are present, the immature cells seen are

5 significant for this disorder, especially the blasts. Therefore, while classifying the blasts as myeloid in origin was not determined by their morphology, identifying the immature cells as blasts was the first step in the diagnostic process. The laboratory professional plays an important role in quality patient care by properly identifying cells. ASCP 2010